The present invention relates to a cartilage regeneration and repair product that induces cell ingrowth into a bioresorbable material and cell differentiation into cartilage tissue, and to methods of using such a product to repair cartilage lesions.
Articular cartilage, an avascular tissue found at the ends of articulating bones, has limited natural capacity to heal. During normal cartilage ontogeny, mesenchymal stem cells condense to form areas of high density and proceed through a series of developmental stages that ends in the mature chondrocyte. The final hyaline cartilage tissue contains only chondrocytes that are surrounded by a matrix composed of type II collagen, sulfated proteoglycans, and additional proteins. The matrix is heterogenous in structure and consists of three morphologically distinct zones: superficial, intermediate, and deep. Zones differ among collagen and proteoglycan distribution, calcification, orientation of collagen fibrils, and the positioning and alignment of chondrocytes (Archer et al., 1996, J. Anat. 189(1):23-35; Morrison et al., 1996, J. Anat. 189(1): 9-22; and Mow et al.,1992, Biomaterials 13(2): 67-97). These properties provide the unique mechanical and physical parameters to hyaline cartilage tissue.
The meniscus, a C-shaped cartilaginous tissue, performs several functions in the knee including load transmission from the femur to the tibia, stabilization in the anterior-posterior position during flexion, and joint lubrication. Damage to the meniscus results in reduced knee stability and knee locking. Over 20 years ago, meniscectomies were performed which permitted immediate pain relief, but were subsequently found to induce the early onset of osteoarthritis (Fairbank, J. Bone Joint Surg. 30B: 664-670; Allen et al., 1984, J. Bone Joint Surg. 66B:666-671; and Roos et al., 1998, Arth. Rheum. 41:687-693). More recently, partial meniscectomies and repair of meniscal tears have been performed (FIGS. 9A-D; Jackson, D., ed., 1995, Reconstructive Knee Surgery Master Techniques in Orthopedic Surgery, ed. R. Thompson, Raven Press: New York). However, partial resection results in the loss of functional meniscus tissue and the early onset of osteoarthritis (Lynch et al., 1983, Clin. Orthop. 172:148-153; Cox et al., 1975, Clin. Orthop. 109:178-183; King, 1995, J. Bone Joint Surg. 77B:836-837). Additionally, repair of meniscal tears is limited to tears in the vascular ⅓ of the meniscus; tears in the semivascular to avascular ⅔ are not repairable (FIGS. 9A-D; Jackson, ibid.). Of the approximately, 560,000 meniscal injuries that occur annually in the United States, an estimated 80% of tears are located in the avascular, irreparable zone. Clearly, a method that both repairs xe2x80x9cnon-repairablexe2x80x9d tears or that can induce regeneration of resected menisci would be valuable for painless musculoskeletal movement and prevention of the early onset of osteoarthritis in a large segment of the population.
The proximal, concave surface of the meniscus contacts the femoral condyle and the distal, flat surface contacts the tibial plateaus. The outer one-third of the meniscus is highly vascularized and contains dense, enervated, connective tissue. In contrast, the remaining meniscus is semivascular or avascular, aneural tissue consisting of fibrochondrocytes surrounded by abundant extracellular matrix (McDevitt et al., Clin. Orthop. Rel. Res. 252:8-17). Fibrochondrocytes are distinctive in both appearance and function compared to undifferentiated fibroblasts. Fibroblasts are elongated cells containing many cellular processes and produce predominantly type I collagen. The matrix produced by fibroblasts does not produce a sufficient mechanical load. In contrast, fibrochondrocytes are round, and are encompassed by lacunae that consists of type I and type II collagen and proteoglycans. These matrix components support compressive forces that are commonly exerted on the meniscus during musculoskeletal movement.
In the 1960""s, demineralized bone matrix was observed to induce the formation of new cartilage and bone when implanted in ectopic sites (Urist, 1965, Science 150:893-899). The components responsible for the osteoinductive activities were termed Bone Morphogenetic Proteins (BMP). At least seven individual BMP proteins were subsequently identified from bone (BMP 1-7) and amino acid analysis revealed that six of the seven BMPs were related to each other and to other members of the TGF-xcex2 superfamily. During endochondral bone formation, TGF-xcex2 family members direct a cascade of events that includes chemotaxis, differentiation of pluripotential cells to the cartilage lineage, maturation of chondrocytes to the hypertrophic stage, mineralization of cartilage, replacement of cartilage with bone cells, and the formation of a calcified matrix (Reddi, 1997, Cytokine and Growth Factor Reviews 8:11-20). Although individual, recombinant BMPs can induce these events, the prevalence of multiple TGF-xcex2 family members in bone tissue underlies the complexity involved in natural osteogenesis.
Bone Protein (Sulzer Orthopedics Biologics, Denver, Colo.), also referred to herein as BP, is a naturally derived mixture of proteins isolated from demineralized bovine bones that has osteogenic activity in vitro and in vivo. In the rodent ectopic model, BP induces endochondral bone formation or bone formation through a cartilage intermediate (Damien, C. et al., 1990, J. Biomed. Mater. Res. 24:639-654). BP in combination with calcium carbonate promotes bone formation in the body (Poser and Benedict, PCT Publication No. WO95/13767). In vitro, BP has been shown to promote differentiation to cartilage of murine embryonic mesenchymal stem cells (Atkinson et al., 1996, In xe2x80x9cMolecular and Developmental Biology of Cartilagexe2x80x9d, Bethesda, Md., Annals New York Acad. Sci. 785:206-208; Atkinson et al., 1997, J. Cell. Biochem. 65:325-339) and of adult myoblast and dermal cells (Atkinson et al., 1998, 44th Annual Meeting, Orthopaedic Research Society, abstract). To ensure chondrogenesis in these in vitro systems, however, culture conditions must be tightly controlled throughout the culture period, including by controlling cellular organization within the culture, optimizing media formulations, and adding exogenous factors that must be carefully established to maximize chondrogenesis over mitogenesis. Such optimization of conditions makes the application of the disclosed in vitro methods to an in vivo system unrealistic and unpredictable. In addition, although in vitro cultures of adult myoblast and dermal cells initially resulted in chondrogenesis, the effect was only transient and over time, the cultures reverted to their original phenotype. Although certain embryonic and precursor cell types showed prolonged chrondrogenesis in vitro in these studies, it would be unpredictable or even impossible in the case of embryonic cells that these specific cell types could be recruited to a site in vivo in an adult patient.
Atkinson et al., in PCT Application No. PCT/EP/05100, incorporated herein by reference in its entirety, describe a delivery system for osteoinductive and/or chondroinductive mixture of naturally derived factors for the induction of cartilage repair.
Hunziker (U.S. Pat. Nos. 5,368,858 and 5,206,023) describes a cartilage repair composition consisting of a biodegradable matrix, a proliferation and/or chemotactic agent, and a transforming factor. A two-stage approach is used where each component has a specific function over time. First, a specific concentration of proliferation/chemotactic agent fills the defect with repair cells. Second, a larger transforming factor concentration, preferably provided in conjunction with a delivery system, transforms repair cells to chondrocytes. The second stage delivery of a high concentration of transforming factor in a delivery system (i.e., liposomes) was required to obtain formation of hyaline cartilage tissue at the treatment site.
Chen and Jeffries (U.S. Pat. No. 5,707,962) describe osteogenic compositions consisting of collagen and sorbed factors to enhance osteogenesis.
Valee and King (U.S. Pat. No. 4,952,404) describe healing of injured, avascular meniscus tissue by release of the angiogenic factor, angiogenin, over at least 3 weeks.
Previously, Amoczky et al. described a method using an autogenous fibrin clot to repair an avascular, circular lesion in canine menisci (Amoczky et al., 1988, J. Bone Joint Surg. 70A:1209-1217). This approach enhanced repair of meniscal tissue compared to controls lacking the fibrin clot. However, the repair tissue was not meniscus-like tissue, but rather connective scar tissue.
Hashimoto et al. described a method using fibrin sealant with or without endothelial cell growth factor in avascular, circular meniscal defects in the canine model (Hashimoto et al., 1992, Am. J. Sports Med. 20:537-541). The growth factor added a modest benefit compared to healing with fibrin sealant alone and this additional effect was not observed until three months after treatment, indicating an indirect contribution of the growth factor. In addition, the defect was filled with hyaline cartilage-like cells, which are not typically present in normal meniscus tissue.
Shirakura, et al. describe the use of an autogenous synovium graft sutured into meniscal tears. While the synovium did enhance healing in ⅓ of the animals, the grafts healed with fibrous tissue, not fibrocartilaginous tissue normally observed in meniscus tissue (Shirakura, 1997, Acta. Orthop. Scand. 68:51-54). Furthermore, ⅔ of the grafts did not heal.
The molecular mechanism for cartilage and bone formation has been partially elucidated. Both bone morphogenetic proteins (BMP) and transforming growth factor xcex2 (TGFxcex2) molecules bind to cell surface receptors (i.e., TGFxcex2/BMP receptors) to initiate a cascade of signals to the nucleus that promotes proliferation, differentiation to cartilage, and/or differentiation to bone (Massague, 1996, Cell 85:947-950). In 1984, Urist described a substantially pure, but not recombinant, BMP combined with a biodegradable poly(lactic acid)polymer delivery system for bone repair (U.S. Pat. No. 4,563,489). This system blends together equal quantities of BMP and poly(lactic acid) (PLA) powder (100 xcexcg of each) and decreases the amount of BMP required to promote bone repair.
Hattersley et al. (WO 96/39170) disclose a two factor composition for inducing cartilaginous tissue formation using a cartilage formation-inducing protein and a cartilage maintenance-inducing protein. Specific recombinant cartilage inducing proteins are specified as BMP-13, MP-52 and BMP-12, and specific cartilage maintenance-inducing proteins are specified as BMP-9. In one embodiment, BMP-9 is encapsulated in a resorbable polymer system and delivered to coincide with the presence of cartilage formation inducing protein(s).
Laurencin et al. (U.S. Pat. No. 5,629,009) disclose a chondrogenesis-inducing device, consisting of a polyanhydride and polyorthoester, that delivers water soluble proteins derived from demineralized bone matrix, TGFxcex2, epidermal growth factor (EGF), fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF).
Previously, Li and Stone (U.S. Pat. No. 5,681,353) have described a Meniscal Augmentation Device that consists of biocompatible and bioresorbable fibers that acts as a scaffold for the ingrowth of meniscal fibrochondrocytes, supports normal meniscal loads, and has an outer surface that approximates the natural meniscus contour. After partial resection of the meniscus to the vascular zone, this device is implanted into the resulting segmental defect. The results have been described in both canines and humans (Stone et al., 1992, Am. J. Sports Med. 20:104-111; and Stone et al., 1997, J. Bone Joint Surg. 79:17701777).
The Meniscus Augmentation Device, the research reports described above, and current repair surgeries provide encouraging results in the area of cartilage repair, but are not satisfactory to induce repair of xe2x80x9cnon-repairablexe2x80x9d avascular tears in which the repair tissue is meniscus tissue, and are not satisfactory to produce short patient rehabilitation times and regenerated meniscus tissue in the vascular zone. Furthermore, no reports have been described in which demonstrate enhanced healing rates of xe2x80x9crepairablexe2x80x9d meniscal tears in vivo.
The present invention relates to a product and method for repairing and/or regenerating cartilage lesions. The product and method of the present invention are useful for repairing a variety of cartilage lesions, including articular and mensical lesions, including vascular, semivascular and avascular lesions. Moreover, the product and method of the present invention can be used to repair different sizes and shapes of cartilage lesions, including radial tears, bucket handle tears, and segmental defects.
One embodiment of the present invention is directed to a product for repair of cartilage lesions. The product includes: (a) a cartilage repair matrix; and (b) a cartilage-inducing composition associated with the matrix for provision of chondrogenesis-enhancing proteins. Each of the chondrogenesis-enhancing proteins can be provided as a protein and/or by a recombinant nucleic acid molecule encoding the protein, such recombinant nucleic acid molecule being operatively linked to a transcription control sequence, and mixtures of proteins and recombinant nucleic acid molecules can be provided. The mixture of chondrogenesis-enhancing proteins include at least a first and second transforming growth factor xcex2 (TGFxcex2) superfamily protein, wherein the first and second TGFxcex2 superfamily protein are different, and at least one protein from a family of proteins selected from growth factor proteins and bone matrix proteins. The chondrogenesis-enhancing proteins are further characterized in that, when cultured together with ATDC5 cells for seven days at a concentration of about 100 ng/ml or less, induce a statistically significant increase in A595 in an Alcian Blue assay performed with said cells.
The product of the present invention can also be formulated to include: (a) a cartilage repair matrix; and (b) a cartilage-inducing composition associated with the matrix, which includes cells that have been cultured with the above-described mixture of chondrogenesis-enhancing proteins.
The cartilage repair matrix of a shape and size that conforms to the cartilage defect such that the defect is repaired. As such, the matrix can be configured as a sheet, which is most suitable for repairing cartilage tears, or the matrix can be configured to repair a segmental defect, which can include a tapered shape. The cartilage repair matrix can be formed of any suitable material, including synthetic polymeric material and ground substances. In one embodiment, the matrix is bioresorbable. In another embodiment, the matrix is porous. When the matrix is configured as a sheet, the matrix is preferably not cross-linked, and when the matrix is configured to repair a segmental defect, the matrix is preferably cross-linked.
The cartilage-inducing composition can be associated with the matrix by any suitable method, including, but not limited to freeze-drying the composition onto a surface of said matrix and suspension within said cartilage repair matrix of a delivery formulation containing said composition. Additionally, the composition can be associated with the matrix ex vivo or in vivo. In one embodiment, a suitable delivery formulation includes nanospheres, wherein the nanospheres are polymer particles having a size of less than 1000 nm and being loaded with between 0.001% and 17% by weight of the cartilage-inducing composition. The nanospheres have an in vitro analytically determined release rate profile with an initial burst of about 10% to about 20% of the total amount of the composition over a first 24 hour period and a long time release rate of a least 0.1% per day during at least seven following days. Yet another suitable delivery vehicle includes a liposome. When the chondrogenesis-enhancing proteins are provided as recombinant nucleic acid molecule, the recombinant nucleic acid molecule can be provided in any suitable form for delivery, including as naked DNA, transformed into a recombinant cell or provided in the form of a recombinant virus.
In preferred embodiments, the TGFxcex2 superfamily proteins included in the chondrogenesis-enhancing protein mixture include, but are not limited to, TGFxcex21, TGFxcex22, TGFxcex23, bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived morphogenetic protein (CDMP)-1, CDMP-2, and/or CDMP-3; and more preferably include TGFxcex21, TGFxcex22, TGFxcex23, bone morphogenetic protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, and/or a cartilage-derived morphogenetic protein (CDMP). The bone matrix proteins included in the chondrogenesis-enhancing protein mixture include, but are not limited to, osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase, cathepsin L pre, osteopontin, matrix GLA protein (MGP), biglycan, decorin, proteoglycan chondroitin sulfate-III (PG-CS III), bone acidic glycoprotein-75 (BAG-75), thrombospondin (TSP) and fibronectin; and more preferably include osteocalcin, osteonectin, MGP, BSP, lysyloxidase and cathepsin L pre. The growth factor proteins included in the chondrogenesis-enhancing protein mixture include, but are not limited to, fibroblast growth factor I (FGF-I), FGF-II, FGF-9, leukocyte inhibitory factor (LIF), insulin, insulin growth factor I (IGF-I), IGF-II, platelet-derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2 (SDF-2), pituitary thyroid hormone (PTH), growth hormone, hepatocyte growth factor (HGF), epithelial growth factor (EGF), transforming growth factor-xcex1 (TGFxcex1) and hedgehog proteins; and more preferably include at least fibroblast growth factor I (FGF-I). The chondrogenesis-enhancing proteins can also include one or more serum proteins, including, but not limited to albumin, transferrin, xcex12-Hs GlycoP, IgG, xcex11-antitrypsin, xcex22-microglobulin, Apo A1 lipoprotein (LP) and Factor XIIIb; and more preferably include albumin, transferrin, Apo A1 LP and Factor XIIIb. Particularly preferred mixtures of chondrogenesis-enhancing proteins include TGFxcex21, TGFxcex22, TGFxcex23, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, and cathepsin L pre, and in another embodiment, TGFxcex21, TGFxcex22, TGFxcex23, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, MGP, BSP, lysyloxidase, cathepsin L pre, albumin, transferrin, Apo A1 LP and Factor XIIIb. Yet another preferred mixture of chondrogenesis-enhancing proteins are bone protein (BP).
Another embodiment of the present invention relates to a method for repair of cartilage lesions, which includes the steps of implanting and fixing into a cartilage lesion a cartilage repair product of the present invention, as described above. The method of the present invention can be used to enhance the rate and/or quality of repair of vascular cartilage tears and segmental defects, and can provide the ability to repair semivascular and avascular tears and segmental defects that, prior to the present invention, were typically considered to be irreparable. When the lesion is in semivascular or avascular cartilage, the product can additionally include a time controlled delivery formulation.
In one aspect, the method of the present invention includes the use of two cartilage repair products to repair a segmental defect. The first product includes a cartilage repair matrix, which is configured as a sheet, is associated with the chondrogenesis-enhancing proteins as described above. The second product includes a cartilage repair matrix configured to replace cartilage removed from the segmental defect, which may or may not be associated with the chondrogenesis-enhancing proteins of the present invention.